The use of protein tagging to facilitate detailed characterization of target proteins has not only
revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the
protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed
protein characterization is widespread in lower organisms that allow for efficient homologous
recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now
within reach for most experimental systems, including mammalian cell lines cultures. In this work, we
describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification
of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile
Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model
organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background.
By combining these ’Proteotypic peptides for Quantification by SRM’ (PQS peptides) with epitope tags,
we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or
after introduction of these tags in the endogenous proteins through genome engineering. Endogenous
protein tagging for absolute quantification provides a powerful extra dimension to protein analysis,
allowing the detailed characterization of endogenous proteins.

The attached file contains an excel document that consists of 2 worksheets.Sheet 1 clarifies which Skyline library was used for each figure.Sheet 2 is a legend for each Skyline file showing which replicate corresponds to a certain sample (e.g. which constructs were transfected, what cell line was used, used compound, which affinity sequence was used for pullout experiments, ...)

The skyline file "pfu_2016-01-21_09-10-33.sky.zip" present in the section "Targeted MS Runs" contains all results of the pyrococcus furiosus discovery runs.

The dat files resulting from the mascot searches are embedded as a spectral library, whereas the raw files are imported as result files. In this way all peptides can be visualized in the raw files together with the library match.